Fatty Acid Photodecarboxylase Is an Interfacial Enzyme That Binds to Lipid–Water Interfaces to Access Its Insoluble Substrate

Abstract

Fatty Acid Photodecarboxylase (FAP), one of the few natural photoenzymes characterized so far, is a promising biocatalyst for lipid-to-hydrocarbon conversion using light. However, the optimum supramolecular organization under which the fatty acid (FA) substrate should be presented to FAP has not been addressed. Using palmitic acid embedded in phospholipid liposomes, phospholipid-stabilized microemulsions and mixed micelles, we show that FAP displays a preference for FAs present in liposomes and at the surface of microemulsions. Adsorption kinetics onto phospholipid and galactolipid monomolecular films further suggests the ability of FAP to bind to and penetrate into membranes, with higher affinity in the presence of FAs. FAP structure reveals a potential interfacial recognition site with clusters of hydrophobic and basic residues surrounding the active site entrance. The resulting dipolar moment suggests the orientation of FAP at negatively charged interfaces. These findings provide important clues for the mode of action of FAP and the development of FAP-based bioconversion processes. ASSOCIATED CONTENT Supporting InformationThe Supporting Information is available free of charge on the ACS Publications website.Experimental procedures, Supplementary figures, Protein sequences as described in the text. (PDF)

Photobiocatalytic Strategies for Organic Synthesis

“…This assessment agrees with a previous study by Aselmeyer et al which shows that FAP displays its highest activity on ionized fatty acids present in organized lipid assemblies like microemulsions or large unilamellar vesicles compared to bulk fatty acids. 46 They also proposed an interfacial recognition site on FAP located at the entrance of the hydrophobic channel of the enzyme.…”

Autocatalytic effect boosts the production of medium-chain hydrocarbons by fatty acid photodecarboxylase

“…To address this question, we compared the in vitro activity (chemical yield of decarboxylation) of purified Cv FAP on C16 FA (one of the best FAP substrates described so far) ( 1 , 4 ) and C8 FA (the substrate showing the most pronounced autocatalytic effect in this study). Given that FAP is an interfacial enzyme ( 16 ), the saturation of the enzymatic activity for FAs of various chain lengths may occur at very different pH values andsubstrate concentrations, because these parameters strongly influence the structuring of FAs ( 17 ) and thereby the activity of the enzyme. So far, all FAP activity comparisons on different substrates were done essentially at one pH value and with the same substrate concentration.…”

“…The coding sequence corresponding to C. variabilis NC64A FAP was cloned into a pLIC07 plasmid (pLIC07 is a pET-28–based expression vector containing downstream FAP gene, a histidine-tagged thioredoxin). The version of Cv FAP used here is FAPv2, which corresponds to the full-length protein truncated by the first 75 amino acids at the N terminus ( 13 , 16 ). The plasmid pLIC07FAP was then transformed into a BL21 E. coli expression strain harboring the pRIL plasmid that allows the synthesis of the rare transfer RNAs that are not naturally produced in E. coli cells.…”

Using Enzymes to Harvest Fatty Acids from Galactosyldiacylglycerols, the Most Abundant Lipids in Plant Biomass

“…37 Producing FFA with galactolipases could be another way forward. 38 An interesting feature of galactolipids is their natural enrichment in long-chain polyunsaturated FA (PUFA) such as the essential ALA (18:3 n-3) which usually accounts for more than 60% of total FA in galactolipids from plant photosynthetic membranes. 39 This was confirmed here with ALA being the main FA released from spinach, parsley, lettuce, grass, and sugar cane leaves (Figures 4B and 5B) and converted into esters in the presence of alcohols (Figure 8).…”